Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis pathway of the catecholamine neurotransmitters: dopamine, epinephrine and norepinephrine. These neurotransmitters are among the most widely used and universally distributed neurochemical systems in the brain. Imbalances in brain catecholamine levels are associated with several diseases such as Alzheimer's disease, Parkinson's disease, Tourette's syndrome, schizophrenia, and clinical depression (Stull et al., 1996). Because the brain catecholamine levels are directly related to levels of biosynthesis of catecholamines, it is useful to produce neurons which express the rate limiting enzyme in catecholamine biosynthesis, TH. These TH positive neurons can be transplanted into catecholamine deficient patients to provide catecholamines, and they are also useful in the studies of catecholamine biosynthesis and neuronal differentiation.
For example, Parkinson's disease is an age-related disorder characterized by a loss of dopamine neurons in the substantia-nigra of the midbrain, which normally send signals to the basal ganglia using dopamine as the neurotransmitter. The symptoms of Parkinson's disease include tremor, rigidity and ataxia. The disease is progressive but can be treated by administration of pharmacological doses of the precursor for dopamine, L-DOPA. However, with chronic use of pharmacotherapy the patients often become refractory to the continued effect of L-DOPA (Marsden et al., 1977). There are many suggested mechanisms for the development of the refractory state, but the simplest is that the patients reach a threshold of cell loss, wherein the remaining cells cannot synthesize sufficient dopamine from the dopamine precursor. At this stage, TH positive neurons could be transplanted into the patient to compensate for the cell loss.
Since neurons are not capable of replicating, it is not practical to generate the required quantity of TH expressing neurons by culturing neurons in vitro. A good approach would be to culture a precursor cell which can replicate and produce neurons in vitro. Ideally, the precursor cells will proliferate or differentiate in response to different signals such that one can control the quantity and timing of neuron production.
Multipotent neural stem cells are an ideal precursor cell for this purpose. Methods of isolating and culturing multipotent neural stem cells in vitro have previously been developed (for example see U.S. Pat. Nos. 5,750,376; 5,980,885; 5,851,832). Briefly, these stem cells may be isolated from both fetal and adult brains, and cultured in vitro indefinitely. These cells retain the ability to proliferate in response to growth factors, or differentiate into all lineages of neural cells (neurons and glia cells, including astrocytes and oligodendrocytes) in response to differentiation stimuli. These cells thus provide an excellent source of neuron production as well as a model system for the study of regulatory mechanisms for neuron production.
Alternatively, it is also desirable to produce TH positive neurons in vivo to compensate for lost or dysfunctional neurons. Accordingly, the need exists for methods of producing TH positive neurons in vitro and in vivo.